Background: A common feature of inflammatory disease is the central role of Fc receptors and immune complexes and the high levels of cytokines produced, which serve to recruit other immune cells thus amplifying the response. Understanding how Fc receptors communicate intracellular signals to initiate cytokine responses and how the released cytokines contribute to inflammation is the major focus of our efforts. Our previous efforts have identified several proteins that are important in the regulation of gene expression initiated by IgE Fc receptor stimulation. We have focused on signaling proteins that may serve as possible links from Fc receptor to gene expression and mast cell degranulation. Our studies demonstrated the functional role of adaptor molecules like the linker for activation of T cells (LAT) and the Grb2-associated binder (Gab2). In the course of these studies we discovered the the IgE Fc receptor utilizes a second receptor-proximal Src family kinase for signal initiation. Fyn kinase was found to be essential for mast cell effector responses and some of its function is mediated through its role in phosphorylating Gab2 leading to activation of phosphatidylinositol 3-OH kinase (PI3K). These findings have provided a new paradigm in how activating Fc receptors couple to signal generation, which is essential for a cells response. Objectives: In the past year our objectives were as follows: 1. To investigate whether receptor proximal kinases play a positive or negative regulatory role in promoting mast cell function. We engaged in this topic of investigation based on the observation that genetic deletion of Lyn kinase results in a hyper-responsive mast cell whereas genetic deletion of Fyn kinase causes hypo-responsiveness. 2. To investigate whether Lyn association with the IgE Fc receptor beta chain has both positive and negative consequences in mast cell effector responses. This line of investigation was based on our prior finding that Lyn phosphorylates multiple intracellular proteins that could function to both augment or inhibit mast cell responses. 3. To investigate the importance of sphingosine kinase in mast cell function. These studies were initiated based on a prior study demonstrating a role for this kinase in mast cell function. Results: The objectives of the past year were met in the following manner. First, our studies on the role of receptor proximal kinases revealed that Lyn kinase has a primary role as a negative regulator of mast cell responsiveness. We found that mice deficient in Lyn showed an allergic-like phenotype that mirrored atopic allergic disease by increased IgE production and increased expression of IgE Fc receptors on the surface of the mast cell. The studies also revealed that the mast cells from these mice were hyper-responsive due in part to increased Fyn kinase activity because the regulation of the activity of this kinase depended on the presence of Lyn. By generating Lyn and Fyn double-deficient mice we found that Fyn functions as the positive driving force for mast cell degranulation. Our studies on the role of the IgE Fc receptor beta chain in regulating mast cell function revealed that the function of this subunit is to amplify mast cell degranulation but also to negatively control mast cell cytokine production. These studies revealed that loss of SHIP-1 (a lipid phosphatase) phosphorylation, due to the loss of Lyn association with receptor, is the likely defect that causes increased NF kappa B activation and cytokine production. Finally, our studies on the role of sphingosine kinase demonstrated that the activation of this kinase upon IgE Fc receptor activation is a requisite step for normal mast cell degranulation and chemotaxis. We found that this is due to the transactivation of a G-protein coupled family of receptors (S1P receptors), which are engaged as a consequence of sphingosine-1-phosphate (S1P) production by activated sphingosine kinase. This work revealed a unique mechanism for augmenting IgE-dependent mast cell function through transactivation of G-protein coupled receptors. Conclusions and Significance: In summary, we found that Lyn kinase functions primarily as a negative regulator of mast cell responsiveness whereas Fyn kinase functions as a positive regulator whose activity is essential to mast cell function. Our studies also point to a requirement of Lyn in controlling B cell mediated IgE production and demonstrate that the association of Lyn with the IgE Fc receptor is important in negative regulation of cytokine production. Loss of this interaction resulted in only partial inhibition of mast cell degranulation demonstrating a role for other signaling molecules. Our findings point to Fyn kinase as the key positive regulator since its genetic deletion in the presence or absence of Lyn causes loss of mast cell degranulation. Our studies have also identified a complementary family of receptors whose transactivation following IgE Fc receptor stimulation is important for full mast cell responses and chemotaxis. In the coming year, we will focus on further understanding the role of Fyn and Lyn kinase in mast cell function with particular emphasis as to whether they play a role in human allergic and autoimmune disease. We also will further explore the importance of sphingosine kinases in immune cell function since the production of S1P has been demonstrated to be enhanced in several diseases including asthma.